Physiology of bacteria in anaerobic environments

Heterotrophic bacteria growing in anaerobic environments face two related problems. First, metabolism of carbon is less efficient without oxygen because cells must get energy through fermentation rather than aerobic respiration. As a result, cells have to either find ways to uptake more carbon or reduce the inefficiency caused by fermentation. Second, these fermentation waste products inhibit the growth of these bacteria. The human gut is an environment that is low in oxygen, yet bacteria thrive and grow to high densities. In this dissertation, I study the adaptations that gut resident bacteria Bacteroides thetaiotaomicron, and Escherichia coli have developed in order to overcome these problems caused by low oxygen. In chapter 2, we study the tolerance of E. coli to acetate stress. Short-chain fatty acids (SCFAs) accumulate in the mammalian gut and other fermentative environments, inhibiting the growth of many bacteria. The cause of growth inhibition and bacterial strategy resisting SCFA stress are however unclear. Through quantitative physiological study of E. coli under acetate stress complemented by proteomic and metabolomic analysis, we establish that acetate accumulation reduces growth by acting as a “useless metabolite” that excludes other metabolites. This accumulation can be blocked by cytosolic acidification, which however has its own deleterious effect. Strikingly, E. coli acidifies its cytosol to an extent that minimizes the combined effect of acidification and acetate accumulation on cell growth. Tolerance to both stress factors would require numerous cytosolic proteins to improve their activities at acidic pH, resulting in an obligatory tradeoff between SCFA tolerance and fast growth in neutral pH as has been reported for various gut bacterial species. In chapter 3, we study the physiology of gut symbiote, Bacteroides thetaiotaomicron. Bacteroides thetaiotaomicron is a dominant member of the intestinal microbiota of humans and other mammals. One reason for B. theta’s dominance is its abundant machinery for utilizing a large variety of complex polysaccharides as a source of carbon and energy. This machinery is organized into localized clusters of genes that coordinate to breakdown polysaccharides of great complexity. While much knowledge has been developed about the individual genes involved in polysaccharide utilization, a quantitative picture is still incomplete. Here, we present a quantitative model describing the growth of B. theta on single and multiple carbon substrates. We find that a dominant obstacle for B. theta’s growth arises from a constant demand for carbon that is independent of growth. This obstacle is partially overcome by B. theta’s ability to utilize multiple carbon sources with existing polysaccharide utilization machinery can provide this flux without a significant proteome cost. In chapter 4, we study the role of pyrophosphate (PPi) in mediating the energy efficiency of bacteria. PPi is a universally abundant molecule found in all domains of life. Despite its ubiquity, many organisms treat it as a waste product, simply degrading the molecule into phosphate. However, some organisms use PPi as a substitute for the energy-carrying ATP. B. theta has multiple enzymes that fulfill this role. Here, we find that depriving B. theta of PPi significantly decreases its yield, which is consistent with PPi’s role as an energy substitute. However, this benefit of PPi also comes at a cost as we also find increasing PPi concentration, a necessity for using it as an energy currency, reduces the rate of protein synthesis and growth. Such a growth-yield tradeoff provides a reason why many organisms don’t take advantage of the free energy benefit provided by substitution of ATP with PPi, often restricted to organisms that would otherwise have a low yield, such as anaerobic bacteria